The experimental approaches to, and application of, NMR spectroscopy of particularly paramagnetic derivatives of O2 binding myoglobin and hemoglobin will be explored and expanded to elucidate electronic and molecular structural control mechanisms of their ligation and autoxidizability. The ultimate aim is to understand the molecular mechanism of oxygen binding so as to aid in the design of genetically engineered hemoglobin as a blood substitute and to understand the pathological properties of natural mutant hemoglobins. The program continues to develop effective methods for definitive assignments and molecular structural determination of the heme cavity, and will emphasize the development of robust bases for interpreting hyperfine shifts for each of the globin oxidation/spin states in terms of functionally relevant structural determinants. This latter goal will rely on detailed studies on a series of diverse, structurally characterized reference globins, a series of point mutants of these reference globin designed to perturb selected structurally/dynamic properties of the active site, as well as both reference and point mutated globins reconstituted with modified hemes designed to separate the rhombic influences of the vinyls from that characteristic of the globin cavity. The hyperfine shifts in cyanomet globins will be interpreted in terms of the axial His orientation, the His-Fe bond strength as well as distal residue hydrogen bonding to, and steric interaction with, the bound ligand. The correlation between globin induced changes in hyperfine shift and axial His orientation in metglobins will be established, probes to quantitate partial water ligation perfected, and the thermodynamics and dynamics of such water ligation characterized. The nature of the distal interactions that determine the unusual orientation of the major magnetic axis in deoxy globins, as well as the correlation between heme contact shift pattern and both His orientation and the presence of non-ligated distal water will be established. The developed approaches will be used to resolve differences in the solution versus crystal orientation of the distal His in Chironomus Hb, structurally characterize equilibrium intermediates in the thermal denaturation of Aplysia Mb, characterize the sequence alignment, folding topology and identify distal residues in a series of "mini" or "compact" (109-118 residue) globins, characterize the distal H- bonding network in the extremely O2-avid, and on occasion, extremely autoxidative-resistant, trematode globins, and characterize the changes in distal interactions and proximal His-Fe bond strain that accompanies the allosteric transition in ligated HbA.